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Cell Growth & Differentiation Vol. 12, 109-117, February 2001
© 2001 American Association for Cancer Research

Inhibition of Transforming Growth Factor ß Signaling in MCF-7 Cells Results in Resistance to Tumor Necrosis Factor {alpha}: A Role for Bcl-21

Stephen W. Tobin2, Mary Kay Brown, Karen Douville, Drew C. Payne, Alan Eastman and Bradley A. Arrick3

Departments of Medicine [S. W. T., K. D., D. C. P., B. A. A.] and Pharmacology [M. K. B., A. E.], Dartmouth Medical School, Hanover, New Hampshire 03755


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Transforming growth factor ß (TGF-ß) is a multifunctional cytokine capable of regulating diverse cellular processes. In this study we investigated the effect of autocrine TGF-ß signaling on tumor necrosis factor (TNF) {alpha}-induced cell death. We abrogated the TGF-ß autocrine loop by overexpression of a truncated TGF-ß type II receptor in MCF-7 breast carcinoma cells and found that this generated resistance to TNF-{alpha}-induced cytotoxicity. To elucidate the molecular basis of the influence of TGF-ß on TNF-{alpha}-induced cytotoxicity, we evaluated the expression levels or activities of proteins involved in TNF-{alpha} signal transduction or the regulation of apoptosis in general in TGF-ß-responsive and TGF-ß-nonresponsive MCF-7 cells. We observed no significant difference in the expression of TNF-{alpha} receptors or the TNF receptor-associated death domain protein. In addition, downstream activation of nuclear factor {kappa}B by TNF-{alpha} was not altered in cells that had lost TGF-ß responsiveness. Analysis of members of the Bcl-2 family of apoptosis-regulatory proteins revealed that Bcl-XL and Bax expression levels were not changed by disruption of TGF-ß signaling. In contrast, the TGF-ß-nonresponsive cells expressed much higher levels of Bcl-2 protein and mRNA than did cells with an intact TGF-ß autocrine loop. Furthermore, restoration of a TGF-ß signal to MCF-7 cells that had spontaneously acquired resistance to TGF-ß caused a reduction in Bcl-2 protein expression. Taken together, our data indicate that loss of autocrine TGF-ß signaling results in enhanced resistance to TNF-{alpha}-mediated cell death and that this is likely to be mediated by derepression of Bcl-2 expression.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
TGF-ß4 is a multifunctional peptide growth factor that plays a pivotal role in many physiological and pathological processes. The principle effects of TGF-ß include regulation of cell proliferation, cell migration, cellular differentiation, immune cell behavior, and the synthesis and degradation of the extracellular matrix (see Refs. 1 and 2 for a general review of TGF-ß). TGF-ß elicits its effects by binding and activating specific receptors expressed on the surface of cells. The TßR-I and TßR-II are transmembrane serine/threonine kinases directly involved in signal propagation. Because both ligand and receptors are expressed in many different tissues and cell types, TGF-ß contributes to a wide range of biological processes via autocrine and paracrine pathways. The full measure of the potential complexity of the effects of TGF-ß can come into play during the initiation and progression of malignancy. Autocrine effects of TGF-ß have been implicated in some models of carcinogenesis and local invasion, whereas paracrine actions of TGF-ß may facilitate metastatic spread and tumor cell escape from immune surveillance (3 , 4) . Indeed, analysis of the production of TGF-ß by tumor cells, as well as that of the functional status of the TGF-ß signal transduction pathway in tumors, has generated important insights into tumor cell biology.

Among the least understood aspects of TGF-ß biology is its role in regulating apoptosis and cell death. Many groups have documented an increase in TGF-ß mRNA and/or protein as an early event in the apoptotic cascade. Examples of increased TGF-ß production by cells undergoing apoptosis include removal of hormone from hormonally dependent epithelia (5, 6, 7) , antimetabolite-induced apoptosis of hormone-independent tumor cells (8) , and treatment of human mammary carcinomas with pharmacological doses of antiestrogens (9 , 10) . What remains unclear, however, is whether the observed rise in TGF-ß is an integral component of the apoptotic cascade or whether it is simply a consequence of the cytotoxic insult.

An investigation by Danforth and Sgagias (11) attempted to elucidate the role, if any, of TGF-ß as a determinant of cell death induced by TNF-{alpha}. TNF-{alpha} is an inflammatory cytokine capable of binding and activating cell surface receptors (TNF-RI and TNF-RII). TNF-RI is a member of a family of death-inducing receptors that includes Fas/CD95 and death receptor 3. Binding of TNF-{alpha} to TNF-RI is responsible for the generation of many of the known cellular responses of TNF-{alpha}, including apoptosis and activation of NF-{kappa}B (12) . Danforth and Sgagias (11) observed that breast cancer cell lines that were susceptible to TNF-{alpha}-mediated cytotoxicity exhibited an increase in TGF-ß production within 24–48 h after exposure to TNF-{alpha} and before cell death. On coincubation with neutralizing anti-TGF-ß antibody, they observed no suppression of TNF-{alpha}-mediated killing and therefore concluded that TGF-ß played no critical role in this process. However, this experimental strategy cannot rule out the possibility that TGF-ß is involved in autocrine regulation of apoptosis because exogenous antibody may not gain access to all of the TGF-ß capable of eliciting an effect.

A more definitive test of the hypothesis that an autocrine effect of TGF-ß plays an important role in the TNF-{alpha}-mediated apoptotic death of the cell could be accomplished by specific and complete abrogation of the TGF-ß pathway. We therefore transfected MCF-7 cells that are sensitive to both TGF-ß and TNF-{alpha} with a truncated TßR-II. Others have demonstrated that when overexpressed, a truncated TßR-II acts in a dominant negative fashion, resulting in loss of TGF-ß responsiveness (13) . Comparison of the resulting TGF-ß-nonresponsive cells to their TGF-ß-responsive counterparts demonstrated that the autocrine production of TGF-ß sensitizes cells to the cytotoxic effects of TNF-{alpha}. Analysis of TNF-{alpha} signaling and expression of apoptosis-regulating genes in these cells has implicated TGF-ß-mediated repression of Bcl-2 expression as the potential underlying mechanism for our observations.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Truncated TßR-II Functions as a Dominant Negative Inhibitor in MCF-7 Cells.
TGF-ß-responsive MCF-7 cells were transfected with a bicistronic mammalian expression plasmid construct, TREZ, which encodes a truncated TßR-II sequence lacking the cytoplasmic kinase domain and a zeocin resistance gene for selection, or with a control vector plasmid, EZ, which encodes the zeocin resistance gene only. After selection in 50 µg/ml zeocin for 4–6 weeks, zeocin-resistant clones were isolated, and these stable transfectants were screened by Western analysis to verify expression of the truncated TßR-II (Fig. 1)Citation . As expected, the MCF-7/TREZ clones express high levels of truncated TßR-II in addition to the full-length endogenous TßR-II, whereas the MCF-7/EZ clones express only the full-length endogenous receptor. Furthermore, it should be noted that the expression level of the truncated TßR-II in MCF-7/TREZ clones is many fold higher than that of the endogenous receptor, which is important for it to function effectively as a competitive dominant negative inhibitor. The additional higher molecular weight bands found in the MCF-7/TREZ clones, which may have resulted from our use of a bicistronic vector, did not interfere with the desired function of the truncated receptor (see below).



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Fig. 1. All MCF-7 stable transfectants express full-length endogenous TßR-II, but the truncated TßR-II is found only in MCF-7/TREZ clones. Cell lysates were prepared and analyzed by Western blot (50 µg/lane) with anti-TßR-II as the primary antibody. The epitope of this antibody consists of amino acids 246–266, which are present in both the truncated and endogenous receptors. Note that all clones express the full-length receptor at Mr 85,000 and the MCF-7/TREZ clones express the truncated receptor near Mr 38,000.

 
Growth inhibition of epithelial cells provides a convenient and relevant functional assay for measuring TGF-ß sensitivity. Therefore, to evaluate the TGF-ß responsiveness of the MCF-7/TREZ and the MCF-7/EZ stable clones, cell proliferation of each clone was measured in the presence or absence of TGF-ß for 5 days (Fig. 2)Citation . MCF-7/EZ clones retain their TGF-ß responsiveness and display 50% growth inhibition at 1 ng/ml TGF-ß, whereas the MCF-7/TREZ clones showed no growth-inhibitory effect at up to 3 ng/ml TGF-ß. The doubling times of the different clones in the absence of exogeneous active TGF-ß were not significantly different from one another (data not shown), suggesting that insufficient amounts of active TGF-ß are produced under standard growth conditions to exert an autocrine antiproliferative effect. The loss of TGF-ß sensitivity in the MCF-7/TREZ clones indicates that the truncated TßR-II functions as a dominant negative inhibitor of TGF-ß signaling in these cells, as has been documented for mink lung epithelial cells and cardiac myocytes (13 , 14) .



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Fig. 2. Truncated TßR-II functions as a dominant negative mutation and effectively blocks TGF-ß signaling. Cells were counted and plated in a 96-well plate at 3000 cells/well. TGF-ß was added to triplicate wells at doses of 0, 0.3, 1.0, or 3.0 ng/ml, and cells were incubated at 37°C for 5 days. A colorimetric MTS reduction assay was conducted to determine the relative number of viable cells in each well. Each clone served as its own control, and values given are a percentage of the untreated control. SDs of the triplicate samples averaged less than 3.5% of the mean. This experiment was repeated three times with similar results.

 
TGF-ß Signaling Alters TNF-{alpha}-induced Cytotoxicity.
Other investigators have shown that treatment of MCF-7 cells with TNF-{alpha} results in up-regulation of the production and secretion of TGF-ß by the treated cells (11) . To evaluate whether TGF-ß signaling plays a functional role in TNF-{alpha}-induced cytotoxicity, the MCF-7/EZ and MCF-7/TREZ clones were treated with a wide range of TNF-{alpha} concentrations for 5 days (Fig. 3)Citation . The TGF-ß-responsive MCF-7/EZ clones were highly sensitive to the cytotoxic effects of TNF-{alpha}, characterized by an IC50 of 100 units/ml. In contrast, the TGF-ß-nonresponsive MCF-7/TREZ clones were relatively resistant to TNF-{alpha}-induced cell death and displayed an IC50 of 800 units/ml. This 8-fold increase in IC50 indicates that TGF-ß signaling via an autocrine loop does play a role in facilitating TNF-{alpha}-induced cytotoxicity. To confirm that TNF-{alpha} was causing cytotoxicity rather than simply growth arrest, cells were assayed for their viability, as measured by the ability to exclude trypan blue. These experiments verified that TNF was indeed resulting in cell death. Specifically, the viability of the TGF-ß-responsive MCF-7/EZ clones after a 5-day treatment with 1000 units/ml TNF was 28.7 ± 4.7%, whereas the viability of the TGF-ß-nonresponsive MCF-7/TREZ clones was 60.0 ± 7.6% (P = 0.0002). We did not observe the changes in cell shape or chromatin condensation typically associated with apoptosis, consistent with the report of Jänicke et al. (15) that MCF-7 cells lack caspase-3 and therefore do not demonstrate DNA fragmentation or visible morphology changes on TNF killing.



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Fig. 3. TGF-ß signaling plays an active role in TNF-{alpha} induced cytotoxicity. Cells were counted and plated in a 96-well plate at 3000 cells/well. TNF-{alpha} was added to triplicate wells at doses ranging from 0 to 3000 units/ml, and the cells were incubated at 37°C for 5 days. A colorimetric MTS reduction assay was conducted to determine the relative number of viable cells in each well. Each clone served as its own control, and values given are a percentage of the untreated control. The TGF-ß-responsive MCF-7/EZ clones show an average IC50 of 100 units/ml TNF-{alpha}, whereas the TGF-ß-nonresponsive MCF-7/TREZ clones have an average IC50 of 800 units/ml TNF-{alpha}. SDs of the triplicate samples averaged less than 3.5% of the mean. This experiment was repeated three times with similar results.

 
Autocrine TGF-ß Signaling Does Not Affect the Expression Levels of TNF-RI or TRADD.
The TNF-{alpha} cell death pathway is initiated when TNF-{alpha} ligand binds its receptor TNF-RI (p55), resulting in recruitment of TRADD to the cytoplasmic death domain of the receptor (12) . To assess whether TGF-ß signaling affects the expression level of these two proteins, Western analysis was conducted on cell lysates of TGF-ß-responsive MCF-7/EZ clones and TGF-ß-nonresponsive MCF-7/TREZ clones (Fig. 4)Citation . Equal levels of the TNF-RI receptor were observed in all clones, whereas the TRADD levels were nearly equivalent. We have also determined that the clones do not differ significantly with regard to their expression of the second TNF-R, TNF-RII (Fig. 4C)Citation . These data argue that TGF-ß signaling has not significantly altered the expression of these proteins and that the marked difference between the MCF-7/TREZ and the MCF-7/EZ clones in their sensitivity to TNF-{alpha}-induced cytotoxicity cannot be attributed to changes in TNF-RI or TRADD protein expression or to the ratio of TNF-Rs.



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Fig. 4. TGF-ß does not affect expression of TNF-RI (p55) or TRADD. Cell lysates were prepared, and 100 µg of protein per lane were loaded on a 10% SDS-PAGE gel. Western immunoblot analyses were carried out using (A) anti-TNF-RI, (B) anti-TRADD, or (C) anti-TNF-RII as the primary antibody.

 
NF-{kappa}B Is Activated in Response to TNF-{alpha} in Both TGF-ß-responsive MCF-7/EZ Clones and TGF-ß-nonresponsive MCF-7/TREZ Clones.
Having established that TGF-ß signaling has not significantly altered the expression of key proteins involved in the early steps of TNF-{alpha} signaling, we evaluated known mechanisms of resistance to TNF-{alpha}-induced cell death. Many studies have shown that activation of the transcription factor NF-{kappa}B can provide an antiapoptotic signal, thereby promoting cell survival (16, 17, 18) . Moreover, evidence in the literature has established that TGF-ß treatment can inhibit activation of NF-{kappa}B in certain systems (19) . To determine whether TGF-ß is attenuating NF-{kappa}B activity in MCF-7 cells, thereby resulting in enhanced sensitivity to TNF-{alpha}-mediated cell death, NF-{kappa}B activity in nuclear extracts of MCF-7/EZ and MCF-7/TREZ clones after treatment with TNF-{alpha} was measured by gel shift assay (Fig. 5A)Citation . The levels of NF-{kappa}B activation in response to TNF-{alpha} are equivalent in both TGF-ß-responsive and -nonresponsive clones, and the maximum activity appears to be reached within the first hour of treatment. In addition to assaying NF-{kappa}B DNA binding activity by gel shift, the transcriptional activity of NF-{kappa}B was measured using a NF-{kappa}B-responsive luciferase reporter construct (Fig. 5B)Citation . In agreement with the gel shift data, the results of the luciferase reporter assays show that the induction of NF-{kappa}B transcriptional activity in response to TNF-{alpha} treatment is equal in the MCF-7/TREZ and MCF-7/EZ clones. These results indicate that the effect of TGF-ß on TNF-{alpha}-induced cytotoxicity is not mediated by changes in NF-{kappa}B activity. Additionally, these data emphasize that although the TGF-ß-nonresponsive MCF-7/TREZ clones are resistant to the cytotoxic effects of TNF-{alpha}, they are still capable of responding to TNF-{alpha} ligand and that certain branches of the TNF-{alpha} signaling cascade remain as sensitive as they are in the TGF-ß-responsive MCF-7/EZ controls.



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Fig. 5. Autocrine TGF-ß signaling does not alter basal levels or TNF-{alpha} induced levels of NF-{kappa}B activity. A, cells were plated in 6-well plates and treated on the following day with or without TNF-{alpha} at 10,000 units/ml for 1 or 4 h. Activation of NF-{kappa}B was measured by gel shift assay. Nuclear extracts were harvested, and 2 µg of nuclear protein were incubated for 20 min with a radiolabeled double-stranded DNA oligonucleotide containing the consensus {kappa}B binding sequence and then electrophoresed on a nondenaturing 4% polyacrylamide gel. Note: Lane 8 contains one-fifth the amount of nuclear extract analyzed in the other lanes. B, cells were transfected with the NF-{kappa}B-responsive luciferase reporter plasmid p-55Ig{kappa}Luc. The next day, the cells were treated with 0, 100, or 300 units/ml TNF-{alpha} for 20 h and then assayed for luciferase activity. Each bar represents the average of duplicate lysates that were analyzed for luciferase activity in triplicate. SDs of the triplicate samples averaged less than 4% of the mean. Statistical analysis indicates that TNF-induced NF-{kappa}B activation in MCF-7/EZ clones is not significantly different from that in MCF-7/TREZ clones (P > 0.6).

 
Loss of Autocrine TGF-ß Signaling Up-Regulates Expression of Bcl-2 but Has No Effect on Bax and Bcl-XS/L.
It has been shown previously that the Bcl-2 family of proteins is a key regulator of cell death induced by many stimuli and that TGF-ß signaling can alter the expression of Bcl-2 family members in a variety of cell types (20, 21, 22, 23) , although breast cancer cells have not been studied in this regard. To ascertain whether or not TGF-ß is altering the cell’s resistance to TNF-{alpha}-induced cell death by modulating the expression of Bcl-2 family members, Western analysis was conducted on cell lysates of TGF-ß-responsive MCF-7/EZ and TGF-ß-nonresponsive MCF-7/TREZ clones (Fig. 6)Citation . There were no significant differences in the expression levels of Bax and Bcl-X. However, all of the MCF-7/TREZ clones express high levels of Bcl-2, whereas the MCF-7/EZ clones express much lower levels of Bcl-2. Analysis of RNA from the clones for expression of Bcl-2 mRNA showed analogous differences between the MCF/EZ and MCF-7/TREZ cells (Fig. 6, E and F)Citation . These data indicate that autocrine TGF-ß signaling in MCF-7 cells down-regulates Bcl-2 expression, thereby rendering the cells sensitive to TNF-{alpha}-induced cytotoxicity. When the TGF-ß autocrine loop is no longer functional, as in the MCF-7/TREZ clones, high levels of Bcl-2 expression result, leading to increased resistance to TNF-{alpha}-induced cell death.



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Fig. 6. Bcl-2 expression is up-regulated by loss of autocrine TGF-ß signaling in MCF-7 cells. Cell lysates were prepared, and 100 µg of protein were loaded per lane of an SDS-PAGE gel. Western immunoblot analyses were carried out using primary antibodies to various members of the Bcl-2 family of proteins. These data reveal no significant differences in the expression of (A) Bax or (B) Bcl-X. However, in C, a large difference in Bcl-2 expression in MCF-7/EZ versus MCF-7/TREZ clones is identified. D, overexposure of the anti-Bcl-2 blot shown in C allows detection of the low level of Bcl-2 protein expressed in the TGF-ß-responsive MCF-7/EZ clones. Reverse transcription-PCR of Bcl-2 mRNA expression after 32 cycles (E) and 38 cycles (F) demonstrates concordance between Bcl-2 protein and RNA expression levels.

 
Restoration of TGF-ß Signaling Results in Down-Regulation of Bcl-2 Expression.
To further investigate the effect of TGF-ß on Bcl-2 expression in MCF-7 cells, we restored TGF-ß signaling to a subline of MCF-7 cells that had lost its responsiveness to TGF-ß. It has been documented that through prolonged passage, sublines of MCF-7 cells have arisen that are no longer responsive to TGF-ß, often with diminished cell surface expression of TGF-ß receptors (24) . We have found that the MCF-7 cells available from Clontech are not growth inhibited by TGF-ß. Furthermore, these cells are resistant to TNF-{alpha}-mediated cell death and express a level of Bcl-2 comparable to that in the MCF-7/TREZ cells (data not shown). Activation of the TGF-ß signal transduction cascade in these cells was achieved by transfection of a novel expression construct for a chimeric protein in which the cytoplasmic kinase domains of TßR-I and TßR-II are tethered to each other, thereby bypassing any receptor defect (25) . In three separate transient transfections of this construct, in which transfection efficiencies of 30–40% were documented by parallel transfections of an expression plasmid for green fluorescent protein, Bcl-2 protein levels were reduced to 50% of transfected controls, on average (P = 0.027). Fig. 7Citation includes Western analysis data from one such experiment. Although these transient transfection experiments do not indicate the mechanism by which TGF-ß signaling affects Bcl-2 levels, they support the notion that the elevated levels observed in the MCF-7/TREZ clones resulted from derepression of Bcl-2 expression as a consequence of loss of TGF-ß responsiveness.



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Fig. 7. Restoration of TGF-ß signaling results in a decrease in Bcl-2 expression. A TGF-ß-nonresponsive subline of MCF-7 cells was transfected with plasmid pXF1042 or the empty vector control, EZ. The pXF1042 construct expresses a chimeric protein containing the cytoplasmic kinase-containing domains of TßR-I and TßR-II, which has been shown to activate TGF-ß signaling in the absence of ligand (25) . Parallel transfections of an expression plasmid for green fluorescent protein, pEGFP-N3 (Clontech), yielded estimated transfection efficiencies of 30–40%. Protein lysates were harvested 3 days after the transfection, and Bcl-2 expression level was analyzed by Western blot, loading 30 µg of total protein per lane.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Considerable progress has been achieved in recent years toward increasing our understanding of pathophysiological mechanisms of cell death, especially in the elucidation of the principal mediators and regulators of apoptosis. Numerous investigators, in the course of characterizing early changes in gene expression during physiological and drug-induced cell death in various epithelial tissues and cancers, have noted a dramatic upsurge in the expression of TGF-ß (5, 6, 7 , 9 , 26, 27, 28, 29) . This led us to ask whether TGF-ß was an integral component of the killing process and, if so, whether the TGF-ß autocrine pathway of cells was protective or alternatively enhanced cell death under these circumstances. We focused our investigation on the cytotoxicity of TNF-{alpha} toward human breast cancer MCF-7 cells because the kinetics and magnitude of TGF-ß induction after exposure to TNF-{alpha}, but before cell death, have been well documented (11) . The studies reported here serve to highlight and characterize an important role for TGF-ß in TNF-{alpha}-mediated killing, possibly via the repression of Bcl-2 expression.

We tested the hypothesis that TGF-ß signaling was an important feature of TNF-{alpha} killing by stable transfection of MCF-7 cells, which were responsive to both cytokines, with a dominant negative truncated TßR-II, thereby blocking functional signal transduction by TGF-ß. We observed that MCF-7 cells transfected with control vector (MCF-7/EZ clones) retained responsiveness to TGF-ß and susceptibility to TNF-{alpha}-mediated cytotoxicity, whereas the cells transfected with the truncated TGF-ß receptor (MCF-7/TREZ clones) were unresponsive to TGF-ß and had acquired resistance to TNF-{alpha} killing. Our findings differ from those of Danforth and Sgagias (11) , who, in similarly motivated experiments, determined that a neutralizing anti-TGF-ß antibody had no effect on TNF-{alpha}-induced growth inhibition of MCF-7 cells. A logical explanation for this apparent discrepancy is that exogenously added antibody resulted in incomplete neutralization of TGF-ß because it could not gain access to all of the TGF-ß. For instance, some TGF-ß may be sequestered in the compartment between the cells and the culture dish.

NF-{kappa}B is a downstream mediator of many of the biological activities of TNF-{alpha}. After TNF binding to cell surface receptors, NF-{kappa}B is liberated from its binding to I{kappa}B in the cytoplasm and translocates to the nucleus to effect changes in gene expression. Of interest, some of the genes that are induced by NF-{kappa}B in response to TNF are protective of TNF-mediated cytotoxicity (16 , 18 , 30) . Indeed, constitutive activation of NF-{kappa}B has been associated with marked resistance to killing by TNF-{alpha} (17) . We therefore performed experiments examining the induction of NF-{kappa}B activity in response to TNF-{alpha} and observed no differences among our clones with regard to the activation or functional activity of NF-{kappa}B, implicating other mechanisms for the TNF resistance of our TGF-ß-nonresponsive cell clones. It is worth emphasizing that the ability of the MCF-7/TREZ cells to bind TNF-{alpha} and properly transduce its signal was not altered, indicating that the overexpression of the dominant negative TßR-II protein did not disrupt TNF-R function.

Further investigation into the molecular mechanism of our basic observation has identified Bcl-2 as a potential intermediary of the effect of TGF-ß. Two prior groups have evaluated the ability of Bcl-2 to protect breast cancer cells from TNF killing. Jäättelä et al. (31) reported that increased expression of Bcl-2 in MCF-7 cells, achieved by transfection of a Bcl-2 expression construct, resulted in marked resistance to cytolysis by either TNF or Fas. Vanhaesebroeck et al. (32) observed only a modest reduction in susceptibility to TNF-induced cytotoxicity in association with increased expression of Bcl-2. Some of the observed differences may relate to the use of MCF-7 cell variants that are in fact quite distinct in many respects. Indeed, a recent direct comparison of three MCF-7 variants revealed strikingly different degrees of TNF-{alpha}-induced apoptosis, some of which directly correlated with differences in basal expression of Bcl-2 (33) . The importance of clonal variants of MCF-7 cells with regard to responsiveness to TGF-ß has been noted by many investigators and has recently been attributed to defective membrane localization of TßR-II in some nonresponsive clones (34) . Our data suggest that some of the clonal differences reported by others in Bcl-2 expression and perhaps in TNF killing as well reflect underlying differences in TGF-ß responsiveness among MCF-7 variants.

Our observations may also be relevant to recently published studies involving a variety of cell systems, including gliomas, Schwann cells, and endometrial epithelial cells, reporting a synergistic proapoptotic effect of TGF-ß when combined with TNF-{alpha} or Fas (35, 36, 37) . Furthermore, the TGF-ß-inducing effect of agents such as vitamin D may explain reports of their ability to potentiate the cytotoxic effect of TNF-{alpha} (38) . Clearly, however, some cell systems have demonstrated TGF-ß-mediated attenuation of cytotoxicity by TNF-{alpha}, underscoring the fact that complex pathways will likely yield distinct results in different cell types (39 , 40) . Some of the differences in reported effects of TGF-ß on TNF-{alpha} cytotoxicity may derive from cell type-specific effects of TGF-ß on expression of Bcl-2. For instance, down-regulation of Bcl-2 after exposure to TGF-ß has been observed in leukemic cell lines, colonic adenoma cells, ovarian carcinoma cells, and umbilical vein endothelial cells (21, 22, 23 , 41 , 42) . In contrast, TGF-ß has been reported to increase the level of Bcl-2 expression in neuronal cells and rheumatoid synovial cells (20 , 43) . Our observation of concordance between Bcl-2 mRNA and protein expression among our clones indicates that the effect of TGF-ß is not mediated through enhanced Bcl-2 protein turnover or attenuation of translation of the Bcl-2 mRNA. We have transfected our cell clones with a human Bcl-2 promoter construct containing the full 5' untranslated region and more than 2.5 kb of upstream promoter sequence, driving the expression of luciferase (44) . In two experiments, we noted no significant differences in promoter activity among the clones or effect of exogenous TGF-ß on luciferase expression of transfected MCF-7/EZ cells (data not shown). This suggests that the effect of TGF-ß on Bcl-2 expression in MCF-7 cells is either posttranscriptional, i.e., mediated by a destabilization of Bcl-2 mRNA, or involves transcriptional regulatory elements that are not contained within this promoter construct.

Dominant repression of Bcl-2 by TGF-ß may be an important element in a number of physiological processes, including apoptosis of the mammary gland during the immediate postlactational period (26 , 45) . Furthermore, loss of the normal TGF-ß/Bcl-2 axis of repression may have important implications in the development and treatment of some forms of malignancy. Disruption of TGF-ß signaling, either by loss of receptor function (46, 47, 48, 49) or due to defective downstream signal propagation (50, 51, 52) , has been reported in a wide range of cancers. Truncation of TßR-II via somatic mutation is an early and likely determining event in colon carcinogenesis in the setting of the hereditary nonpolyposis colorectal cancer syndrome (53) . Bcl-2 can protect cells from a wide range of injurious assaults, including those mediated by various forms of chemotherapy (54 , 55) . Although investigators have not yet correlated absence of functional TGF-ß signaling with resistance to anticancer therapy, such as might well accompany a presumed derepression of Bcl-2 expression, this consequence of the loss of TGF-ß responsiveness by tumors may represent its most pernicious manifestation.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Plasmid DNA Construction and Isolation.
We used a bicistronic plasmid strategy (56) to obtain stable transfectants overexpressing a truncated TßR-II. To accomplish this, we constructed a plasmid, designated TREZ, that directs transcription of a bicistronic mRNA containing the truncated TßR-II followed by the internal ribosomal entry site of the encephalomyocarditis virus, upstream of the coding sequence of the zeocin resistance selectable marker gene. This bicistronic coding sequence was subcloned into the pRK5 expression vector (Genentech), which uses the cytomegalovirus promoter/enhancer to drive expression. Specifically, the 830-bp NcoI-BglII fragment of the TßR-II cDNA clone H2-3FF (57) was subcloned into the EcoRI-BamHI sites of pRK5. Brand and Schneider (13) have reported that truncation of TßR-II at this BglII site results in the generation of a kinase-deficient dominant negative mutant receptor. The resulting plasmid was cut at XbaI and SalI sites downstream of the truncated TßR-II sequence and used in a three-way ligation for the addition of the 599-bp XbaI-NcoI fragment from LZIN (56) containing the internal ribosomal entry site and the 429-bp NcoI-SalI fragment from pZeoSV (Invitrogen) containing the coding sequence for the zeocin resistance gene. The control plasmid, designated EZ, was created by removal of the truncated receptor sequence from the TREZ plasmid by EcoRI digestion and reclosure by intramolecular ligation. The expression plasmid designated pXF1042, which has been shown to bypass receptor defects and induce ligand-independent TGF-ß signaling in cells, was obtained from Dr. Rik Derynck (University of California, San Francisco, CA; Ref. 25 ). All plasmid DNAs were purified using a DNA column chromatography kit (Qiagen).

Cell Culture and Transfection.
The human breast cancer cell line MCF-7 was obtained from American Type Culture Collection. For the experiments with the pXF1042 plasmid, a clone of MCF-7 cells from Clontech (MCF-7 Tet/Off) was used. Cells were maintained in DMEM:Ham’s F-12 supplemented with 10% fetal bovine serum, 2.5 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C, 5% CO2. To generate stable transfectants overexpressing the truncated TßR-II, MCF-7 cells in 10-cm dishes were transfected with 15 µg of the expression plasmid TREZ or control plasmid EZ by calcium phosphate-mediated transfection (58) . Forty-eight h after transfection, the cells were trypsinized and split into 10-cm dishes with selective media containing 50 µg/ml zeocin (Invitrogen). Zeocin-resistant clones were isolated, expanded, and assayed for expression of the transfected gene by Western blot analysis.

Cell Proliferation and Cytotoxicity Assays.
Cells were trypsinized, counted on a hemocytometer, and then plated in a 96-well plate in triplicate at 3000 cells/well in the same serum-containing medium used for routine cell culture. After 6 h, recombinant human TNF-{alpha} or TGF-ß1 (R&D Systems) was added to each well at various concentrations. The plates were incubated at 37°C, 5% CO2 for 5 days. The relative number of viable cells in each well was determined by MTS reduction assay using the Celltiter 96 kit (Promega) according to the manufacturer’s instructions. In some experiments, cell viability was determined by trypan blue exclusion. Cells were trypsinized and mixed with trypan blue, and the number of blue cells and trypan blue-excluding cells was determined by counting cells on a hemocytometer. Triplicate samples were analyzed.

Gel Shift Assay.
Electrophoretic mobility shift assays of nuclear NF-{kappa}B were conducted as described by Barchowsky et al. (59) . Cells were plated in 35-mm wells in triplicate. The next day, cells were treated with 10,000 units/ml TNF-{alpha} for 1 or 4 h or left untreated. Nuclear extracts were harvested, and protein content was determined by the BCA protein assay (Pierce). The gel shift probe was a double-stranded NF-{kappa}B consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3'; Promega) labeled at the 5' end with 32P (58) . Two µg of nuclear protein were incubated with 2 x 105 cpm of 5'-32P-labeled probe and then resolved by electrophoresis in a 4% acrylamide gel under nondenaturing conditions. The gel was then dried and exposed to autoradiographic film.

Luciferase Reporter Assay.
Cells were plated in 10-cm dishes and transfected with the NF-{kappa}B-responsive p-55Ig{kappa}Luc plasmid (60) by LipofectAMINE (Life Technologies, Inc.). The following day, each 10-cm dish was trypsinized, and the cells were divided equally among the wells of a 6-well plate. Cells were treated in duplicate with 0, 100, or 300 units/ml TNF-{alpha} at 37°C for 20 h. To prepare lysates, cells were washed three times with cold PBS, suspended in 200 µl of lysis buffer (1% Triton X-100, 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, and 1 mM DTT), and then centrifuged at 14,000 x g for 5 min at 4°C. The supernatant was assayed for luciferase activity in triplicate in a luminometer. This assay was conducted in a 96-well plate format, with each well containing 20 µl of lysate, 20 µl of lysis buffer, and 145 µl of assay buffer (25 mM glycylglycine, 15 mM potassium phosphate, 15 mM MgSO4, 4 mM EGTA, 2 mM ATP, and 1 mM DTT) with the addition of 40 µl of luciferin solution (400 µM luciferin and 25 mM glycylglycine) at the time each well was read. Luciferase activity of each sample was normalized to the protein content of the lysates, which was assessed by the BCA protein assay (Pierce).

Western Blot Analysis.
Cells were grown in 10-cm dishes, washed twice with cold PBS, and scraped in 1 ml of PBS containing protease inhibitors (9.5 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM pefabloc). The cells were transferred to a microfuge tube and then centrifuged at 150 x g to pellet the cells. The cell pellet was resuspended in lysis buffer [50 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 0.5% NP40] containing the same protease inhibitors. The lysates were shaken on ice for 20 min, followed by centrifugation at 16,000 x g for 10 min at 4°C to pellet insoluble material. The supernatant was transferred to a microfuge tube and stored at -70°C. Protein concentrations were determined by the BCA assay. Cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in 5% milk in TBS and incubated with primary antibody diluted in 5% milk in TBST. After three washes in 5% milk in TBST, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (Jackson Laboratories) diluted 1:2000, washed three times in 5% milk in TBST, rinsed once in TBS, and then developed by enhanced chemiluminescence (Amersham). All experiments involving Western blot analyses were performed at least three times, and one representative blot was used for the relevant figures.

Antibodies.
The antibodies used were as follows: (a) for TßR-II, L-21 (Santa Cruz Biotechnology; 100 µg/ml; used at 0.1 µg/ml; 1:1000); (b) for TNF-RI, 1995-01 (Genzyme; 1 mg/ml; used at 10 µg/ml; 1:100); (c) for TNF-RII, 1888-01 (Genzyme; 1 mg/ml; used at 10 µg/ml; 1:100); (d) for TRADD, T50320 (Transduction Laboratories; 250 µg/ml; used at 1 µg/ml; 1:250); (e) for Bax, N-20 (Santa Cruz Biotechnology; 100 µg/ml; used at 0.13 µg/ml; 1:750); (f) for Bcl-XS/L, M-125 (Santa Cruz Biotechnology; 200 µg/ml; used at 2 µg/ml; 1:100); (g) for Bcl-2, M0887 (Dako; 320 µg/ml; used at 0.16 µg/ml; 1:2000); and (h) for actin, N350 (Amersham; 300 µg/ml; used at 0.075 µg/ml; 1:4000).

RNA Preparation and Analysis.
Total RNA was prepared from cells using the Trizol reagent according to manufacturer’s instructions (Life Technologies, Inc.), and 2 µg of total RNA were used for synthesis of cDNA using random hexamer primers and Moloney murine leukemia virus reverse transcriptase. Equivalent cDNA synthesis between the clones was confirmed with actin-specific primers. PCR of Bcl-2 cDNA was performed with 0.5 µM primers (5'-ACGACTTCTCCCGCCGCTACC; 5'-GTACAGTTCCAC- AAAGGCATCC) at an annealing temperature of 60°C for 30 s and extension at 72°C for 30 s each cycle. Five µl of reverse transcription reaction were used as template. These primers span an intron and yield a 302-bp product, and no product was obtained if reverse transcriptase was ommited from the cDNA synthesis step.

Statistical Analysis.
Statistical significance of the experimental data was determined using the two-tailed, unpaired Student’s t test. P < 0.05 was considered significant.


    Acknowledgments
 
We thank Dr. Rik Derynck for the pXF1042 plasmid used to express the chimeric protein containing the kinase domains of TßR-I and TßR-II, Dr. Harvey Lodish and colleagues for TßR-II cDNA clone H2-3FF, Dr. John Majors for the LZIN plasmid, Dr. Takashi Fujita for the NF-{kappa}B-responsive p-55g{kappa}Luc plasmid, and Dr. Andrew Zelenetz for the BCL2-promoter construct (pGL2-BCL2). We are also grateful for assistance in the analysis of NF-{kappa}B activation provided by Dr. Aaron Barchowsky.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grants DAMD17-J-4287 and DAMD17-J-4130 from the Department of Defense Breast Cancer Research Program. Back

2 Supported by National Cancer Institute Training Grant CA09658. Back

3 To whom requests for reprints should be addressed, at Dartmouth Medical School, Kellogg Box 0128, Hanover, NH 03755. Phone: (603) 650-1550; Fax: (603) 650-1129; E-mail: Bradley.Arrick{at}dartmouth.edu Back

4 The abbreviations used are: TGF-ß, transforming growth factor ß; TßR-I, TGF-ß type I receptor; TßR-II, TGF-ß type II receptor; TNF, tumor necrosis factor; TNF-R, tumor necrosis factor {alpha} receptor; NF-{kappa}B, nuclear factor {kappa}B; TBS, Tris-buffered saline; TRADD, TNF receptor-associated death domain protein; TBST, TBS and 0.05% Tween 20; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt. Back

Received for publication 8/15/00. Revision received 12/19/00. Accepted for publication 12/20/00.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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